Protonated nucleobase ligands: synthesis, structure and characterization of 9-methyladeninium hexachloroplatinate and pentachloro(9-methyladeninium)platinum(IV)

Na(2)[PtCl(6)] was found to react with (9-MeAH)Cl(.)H(2)O (2) (9-MeA=9-methyladenine) in aqueous solution yielding (9-MeAH)(2)[PtCl(6)](.)2H(2)O (3). The same compound was obtained from hexachloroplatinic acid and 9-methyladenine. Performing this reaction at 60 degrees C, complex formation took place yielding the 9-methyladeninium complex [PtCl(5)(9-MeAH)](.)2H(2)O (4a). An analogous complex, [PtCl(5)(9-MeAH)](.)1/2(18C6)(.)H(2)O (4b, 18C6=crown ether 18-crown-6), was formed in the reaction of aquapentachloroplatinic acid (H(3)O)[PtCl(5)(H(2)O)](.)2(18C6)(.)6H(2)O (1) with 9-methyladenine in 1:1 ratio. All complexes were isolated in moderate to good yields as yellow powder (4b) and crystals (3, 4a), respectively. They were fully characterized by microanalysis, IR and NMR ((1)H, (13)C, (195)Pt) spectroscopies, and in part (2, 3, 4a) also by single-crystal X-ray diffraction analysis. Molecular structure of complex 4a exhibited that the 9-methyladeninium ligand is N1 protonated and coordinated through N7 to platinum(IV).     

Chloroplast fructose 1,6-bisphosphatase with changed redox modulation: comparison of the Galdieria enzyme with cysteine mutants from spinach

Spinach fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), a redox-modulated chloroplast enzyme and part of the Calvin cycle, and three different Cys mutants were expressed in E. coli. The properties of the purified proteins were compared to those of native and recombinant chloroplast FBPase from the red alga Galdieria sulphuraria. In spinach chloroplast FBPase, Cys(155) and Cys(174) are engaged in the formation of the disulfide bridge. The corresponding mutants are active when expressed in E. coli, while C179S is inactive and can be reductively activated as can the wild-type enzyme. The active C174S mutant, however, could be inactivated by oxidation, and reactivated, but only by reduction, not alternatively with high pH and high Mg(2+) as is the case for the wild-type enzyme. In the sequence of Galdieria FBPase, the Cys that corresponds to Cys(179) in the spinach enzyme is lacking. However, the Galdieria FBPase, in contrast to the spinach Cys(179) mutant, does not show any indication for a comparable redox modulation of its activity. Instead, oxidation only leads to partial inactivation without any qualitative changes in enzyme properties. Upon reduction, the lost activity can be recovered.     

Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA

The structure of mitochondria is highly dynamic and depends on the balance of fusion and fission processes. Deletion of the mitochondrial dynamin-like protein Mgm1 in yeast leads to extensive fragmentation of mitochondria and loss of mitochondrial DNA. Mgm1 and its human ortholog OPA1, associated with optic atrophy type I in humans, were proposed to be involved in fission or fusion of mitochondria or, alternatively, in remodeling of the mitochondrial inner membrane and cristae formation (Wong, E. D., Wagner, J. A., Gorsich, S. W., McCaffery, J. M., Shaw, J. M., and Nunnari, J. (2000) J. Cell Biol. 151, 341-352; Wong, E. D., Wagner, J. A., Scott, S. V., Okreglak, V., Holewinske, T. J., Cassidy-Stone, A., and Nunnari, J. (2003) J. Cell Biol. 160, 303-311; Sesaki, H., Southard, S. M., Yaffe, M. P., and Jensen, R. E. (2003) Mol. Biol. Cell, in press). Mgm1 and its orthologs exist in two forms of different lengths. To obtain new insights into their biogenesis and function, we have characterized these isoforms. The large isoform (l-Mgm1) contains an N-terminal putative transmembrane segment that is absent in the short isoform (s-Mgm1). The large isoform is an integral inner membrane protein facing the intermembrane space. Furthermore, the conversion of l-Mgm1 into s-Mgm1 was found to be dependent on Pcp1 (Mdm37/YGR101w) a recently identified component essential for wild type mitochondrial morphology. Pcp1 is a homolog of Rhomboid, a serine protease known to be involved in intercellular signaling in Drosophila melanogaster, suggesting a function of Pcp1 in the proteolytic maturation process of Mgm1. Expression of s-Mgm1 can partially complement the Deltapcp1 phenotype. Expression of both isoforms but not of either isoform alone was able to partially complement the Deltamgm1 phenotype. Therefore, processing of l-Mgm1 by Pcp1 and the presence of both isoforms of Mgm1 appear crucial for wild type mitochondrial morphology and maintenance of mitochondrial DNA.     

The urbilaterian brain: developmental insights into the evolutionary origin of the brain in insects and vertebrates

Classical phylogenetic, neuroanatomical and neuroembryological studies propose an independent evolutionary origin of the brains of insects and vertebrates. Contrasting with this, data from three sets of molecular and genetic analyses indicate that the developmental program of brains of insects and vertebrates might be highly conserved and suggest a monophyletic origin of the brain of protostomes and deuterostomes. First, recent results of molecular phylogeny imply that none of the currently living animals correspond to evolutionary intermediates between protostomes and deuterostomes, thus making it impossible to infer the morphological organization of an ancestral bilaterian brain from living specimens. Second, recent molecular genetic evidence provides support for the body axis inversion hypothesis, which implies that a dorsoventral inversion of the body axis occurred in protostomes versus deuterostomes, leading to the inverted location of neurogenic regions in these animal groups. Third, recent developmental genetic analyses are uncovering the existence of structurally and functionally homologous genes that have comparable and interchangeable functions in early brain development in insect and vertebrate model systems. Thus, development of the anteriormost part of the embryonic brain in both insects and vertebrates depends upon the otd/Otx and ems/Emx genes; development of the posterior part of the embryonic brain in both insects and vertebrates involves homologous control genes of the Hox cluster. These findings, which demonstrate the conserved expression and function of key patterning genes involved in embryonic brain development in insects and vertebrates support the hypothesis that the brains of protostomes and deuterostomes are of monophyletic, urbilaterian origin.     

Cytogenetics of three breeds of river buffalo (Bubalus bubalis L.), with evidence of a fragile site on the X chromosome

The cytogenetic study of 182 river buffalo (Bubalus bubalis L., 2n = 50) of Murrah, Mediterranean and Jaffarabadi breeds, from the State of S?o Paulo, was carried out to characterize their chromosomes and to detect possible chromosomal abnormalities. The karyotypes were indistinguishable with conventional staining as well as with C and replication R banding techniques. In about 44% of the sample (8 males and 72 females), an X marker chromosome due to a fragile site was shown. The frequency of metaphases expressing the fragility site on the X was highly variable, from 2.86 to 41.03%. In females, the fragile site, rarely appeared on both X chromosomes. Most of the metaphases showed only 1 marker chromosome. In R-banded metaphases using 5-bromodeoxyuridine (BrdU) treatment, it corresponded in general to the late replicating X chromosome. No correlation between the X fragile site and altered phenotype was found. Structural and numerical chromosome rearrangements were ruled out in the present sample of buffalo.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.      

Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry.

A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol.